The primary cause of cystic fibrosis (CF) is the loss of functional cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent chloride channel. The loss of CFTR function results in a number of manifestations that are not clearly related to cAMP-dependent chloride transport (1). These manifestations include enhanced transepithelial sodium absorption, a propensity for bacterial infection in the lung, and exaggerated inflammatory responses. Recent gene expression array studies demonstrate broad ranging alterations in transcriptional regulation somehow linked to lost CFTR function (2,3). Our laboratory has identified altered expression of individual proteins mostly related to inflammatory signaling in CF epithelial cells including reduced expression of the inducible form of nitric oxide synthase (NOS2) (4-6), reduced expression of Smad3 (7), altered regulation of signal transducer and activator of transcription-1 (Stat1) activity (8), and elevated expression of the small GTPase RhoA (9). A continuing goal of our research is to identify a mechanism related to CFTR function that influences multiple regulatory cascades. Recent evidence in our laboratory suggests many of these signaling alterations are due to CF-related changes in cholesterol processing leading to the hypothesis that lost CFTR function results in impaired cholesterol processing. Our preliminary data demonstrate that CF epithelial cells exhibit an accumulation of unesterified cholesterol compared to wt controls as well as peri-nuclear accumulation of fluorescently labeled cholesterol. Impaired cholesterol transport is a strong candidate for a common regulatory mechanism linking CFTR to multiple cellular functions. Questions raised by these observations are 1) how is CFTR involved in cholesterol processing and 2) what are the consequences of impaired cholesterol transport? To address these questions, we will first determine the nature of altered cholesterol processing in CF cells to understand where in the cholesterol transport system a CF-related alteration is taking place in order to clarify where CFTR function is critical in this process. Secondly, we will determine how cholesterol-dependent regulatory mechanisms are influenced in CF cells to gain a better understanding of the consequences of altered cholesterol regulation. To accomplish these goals, the following specific aims will be pursued: Aim 1. To examine the role of intracellular pH in modulating cholesterol transport. Aim 2. To determine what cholesterol and related lipid transport processes are altered in CF epithelial cells. Aim 3. To examine isoprenoid/cholesterol synthesis and sterol regulatory element binding protein-1 (SREBP-1a and SREBP-1c) and SREBP-2 regulation in CF cells. [unreadable] [unreadable]